CN105807051A - Kit for identifying MTB (mycobacterium tuberculosis) infection and NTM (nontuberculosis mycobacteria) infection - Google Patents
Kit for identifying MTB (mycobacterium tuberculosis) infection and NTM (nontuberculosis mycobacteria) infection Download PDFInfo
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- CN105807051A CN105807051A CN201610210106.3A CN201610210106A CN105807051A CN 105807051 A CN105807051 A CN 105807051A CN 201610210106 A CN201610210106 A CN 201610210106A CN 105807051 A CN105807051 A CN 105807051A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/5695—Mycobacteria
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/35—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycobacteriaceae (F)
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Abstract
The invention discloses a kit for identifying MTB (mycobacterium tuberculosis) infection and NTM (nontuberculosis mycobacteria) infection. The kit comprises an MTB specific antigen, a Mycobacterium specific antigen and a tool for detecting antigen recognition performed by T cells. The kit for identifying the MTB infection and the NTM infection can be used for distinguishing the two kinds of infection, and has higher sensitivity, high specificity and operation safety compared with sputum smears and a sputum culture method; the kit has great guidance significance for doctors in the aspects of clinical diagnosis and medication.
Description
Technical field
The invention belongs to biomedical inspection field, relate to the cellular immunology method of inspection, specifically, relate to a kind of detection kit differentiating that mycobacterium tuberculosis infects with non-tuberculous mycobacteria.
Background technology
Tuberculosis (tuberculosis, TB) it is one of most common chronic infectious disease in worldwide, main by mycobacterium tuberculosis (Mycobacteriumtuberculosis, MTB) infection causes, in addition other mycobacteria especially non-tuberculous mycobacteria (nontuberculosismycobacteria, NTM) constituting about the 5%～15% of clinical case caused by infection, the latter's ratio when acquired immune deficiency syndrome (AIDS) merges tuberculosis is higher.According to statistics, nearly 1/3 population in the whole world has infected Mtb, and wherein the infected of 5%～10% is tuberculosis patient, there are about 8,000,000～900 Wan Xinfa active tuberculosis cases every year, and 2,000,000～3,000,000 is dead because of tuberculosis.
Non-tuberculous mycobacteria (NTM) is other Mycobacterium tuberculosiss in mycobacterium except MTB and Mycobacterium leprae.Finding NTM and 13 subspecies in 154 so far altogether, only human body is cured the disease by small part.Causing common strain sick for NTM is mycobacterium avium-intracellulare complex (Mycobacteriumaviumcomplex or Mycobacteriumavium-intracellularecomplex, MAC), the common chronic respiratory system symptom of infected patient, identical with pulmonary tuberculosis, its clinical manifestation is easily infected the pulmonary tuberculosis caused obscure with MTB, not easily differentiates.And most of NTM is to the conventional anti-equal drug resistance of MTB medicine, for this, in clinic before tuberculotherapy, to MTB and the NTM differentiation infected with identify particularly significant.When traditional method differentiates NTM, it is necessary to after using isolation medium to turn out mycobacterium strain, recycle molecular biological method and identify, such method length consuming time, and detection equipment is required higher, it is unfavorable for Clinical practice.
Therefore, we lack and a kind of quick diagnosis MTB or NTM can infect the effective ways lungy caused.
In addition, MTB infection rate is significantly high, sickness rate is relatively low, but tuberculosis latent infection (latenttuberculosisinfection, LTBI) person reduces at body's immunity or can be changed into tuberculosis patient in other situations, and becomes the source of infection that tuberculosis is dangerous to a great extent together with active tuberculosis patient.LTBI is a kind of sub-clinical state after tubercle bacillus affection, without clinical symptoms, bacteriology and radioscopy often negative, tuberculin skin test (tuberculinskintest, TST) namely purified protein derivative (purifiedproteinderivative, PPD) test can present the positive.But owing to PPD test can not differentiate natural infection and bacillus calmette-guerin vaccine (BCG) inoculation, it is higher that its result may result in MTB infection rate survey result, therefore in the urgent need to researching and developing detection method more special, reliable and reagent.
ELISpot measures (enzymelinkedimmunospotassay, ELISPOT) it is newly-established a kind of cellular immunology detection method in recent years, its ultimate principle is to set up on ELISA method basis, antigen (polypeptide) the specific immune cell release cells factor (such as gamma interferon etc.) can be detected in vitro, it is a kind of based on the immunoreactive Novel immune detection technique of antigen-specific cellular, have been demonstrated (to include bacterium sun and bacterium the moon pulmonary tuberculosis in tuberculosis, the outer tuberculosis of lung) diagnosis, LTBI detects, the aspects such as therapeutic effect monitoring have potential using value.This technology is not affected by BCG inoculation to have research to show, has higher specificity, can significantly improve the verification and measurement ratio of acquired immune deficiency syndrome (AIDS) merging tuberculosis compared with testing with PPD.At present, abroad, based on ELISPOT principle, business-like T-SPOT.TB (OxfordImmunotec, Abingdon, UK), QuantiFERONTB1Gold (QFT-G) and QuantiFERON-TB1GoldIn-tube (QFT-IT) (Cellestis based on ELISA principle, Carnegie, Victoria, Australia) tuberculosis IFN-γ release test is used by multiple state approvals, has been put in many American-European countries in LTBI and tuberculosis contactee's guide detection；But due to expensive and limit and widely use in China, and data show that the detection to Chinese population has relatively low sensitivity, have research prompting this to be likely to, from crowd, the reaction table of different polypeptide fragments is revealed certain race difference relevant；Meanwhile, the tuberculosis polypeptide majority in a few money ELISPOT reagent of China's research is not independent research.Additionally, above-mentioned all reagent is both for MTB infects detection, there is no MTB and the NTM ELISPOT report infecting simultaneously and differentiating detection.
Summary of the invention
In order to overcome prior art Problems existing, the present invention provides a kind of test kit differentiating that mycobacterium tuberculosis infects with non-tuberculous mycobacteria.
The technical solution adopted in the present invention is:
A kind of test kit differentiating that mycobacterium tuberculosis infects with non-tuberculous mycobacteria, including the specific antigen of mycobacterium tuberculosis, mycobacteria genus-specific antigen, and the detection T cell instrument to described antigen recognition.
Further, described test kit is by utilizing the specific antigen of mycobacterium tuberculosis to contact with ex vivo T-cell to be measured respectively with mycobacteria genus-specific antigen, and detect the cytokine that this T cell discharges, identify that testing sample is mycobacterium tuberculosis or non-tuberculous mycobacteria infection.
Further, specific for described mycobacterium tuberculosis antigen and mycobacteria genus-specific antigen testing result are compared, judge that described ex vivo T-cell to be measured is m tuberculosis infection or non-tuberculous mycobacteria infection according to comparative result.
Further, if the specific antigen of described mycobacterium tuberculosis and the detection of mycobacteria genus-specific antigen are all positive, then described ex vivo T-cell to be measured is m tuberculosis infection；If if the specific Detection of antigen result of described mycobacterium tuberculosis is negative, described mycobacteria genus-specific antigen testing result is positive, then described ex vivo T-cell to be measured is that non-tuberculous mycobacteria infects.
Further, the specific antigen of described mycobacterium tuberculosis includes one or more in polypeptide E6, E7, C14.
Further, the instrument of described antigen recognition includes IFN-γ antibody.
Further, the cytokine of the ex vivo T-cell release that the tool detection of described antigen recognition is to be measured.
Further, described T cell derives from blood, bronchoalveolar lavage fluid, hydrothorax, cerebrospinal fluid, lymph node.
Further, described test kit also includes negative control group and positive controls, and described positive controls contains PMA and ionomycin.The invention have the benefit that
The present invention utilizes mycobacteria genus-specific antigen, in conjunction with the ELISPOT detection method of the specific detection of MTB, it is possible to identification of M TB and NTM infects simultaneously.The present invention differentiates the test kit that mycobacterium tuberculosis infects with non-tuberculous mycobacteria, can distinguish both and infect, compare Sputum smears and Sputum culturing method has higher susceptiveness and specificity and processing safety.Clinical diagnosis and medication to doctor have important directive significance.
In order to be more fully understood that and implement, describe the present invention in detail below in conjunction with accompanying drawing.
Accompanying drawing explanation
Fig. 1 is the test kit detection hole differentiating mycobacterium tuberculosis and non-tuberculous mycobacteria infection and control wells setting, the result schematic diagram of the present invention, and wherein, detection 1 arranges MTB specificity E6+E7+C14 mixed polypeptide；Detection 2 arranges Mycobacterium specificity mixed polypeptide；Comparison 1: for positive (PMA+Inomycin) comparison；Comparison 2: negative control；The T-SPOT.TB of T1 and T2 respectively parallel control.
Detailed description of the invention
[embodiment 1] the present embodiment includes a kind of test kit differentiating that mycobacterium tuberculosis infects with non-tuberculous mycobacteria
Refer to Fig. 1, this test kit includes detection hole, control wells and parallel control wells, and detectable therein is set to:
Detection 1:MTB specificity E6+E7+C14 mixed polypeptide
Detection 2: Mycobacterium specificity mixed polypeptide；
Comparison 1: positive control: PMA+Inomycin)；
Comparison 2: negative control: be not added with polypeptide；
The T-SPOT.TB detection of T1 and T2 respectively parallel control.
Detecting step is as follows:
It is standby that 1 tuberculosis patient separating monocytic cell (PBMC) separation is resuspended in R10 culture fluid (containing 10% calf serum in RPMI1640 culture fluid) with Ficoll lymphocyte separation medium separation PBMC, PBMC.
2ELISPOT analyzes and selects band pvdf membrane 96 orifice plate as Sptting plate, is coated overnight by anti-human gamma interferon (IFN γ) monoclonal antibody (mAb), within second day, adds 5 × 105PBMC and corresponding detectable.3rd day, being sequentially added into the anti-human IFN γ mAb of biotin labeling, streptavidin-alkali phosphatase reacts, and BCIP/NBT develops the color.Result reads on ELISPOTReader.With SFC (Spot-formingcells)/106PBMC > 50 is as being judged as the positive.
Result judges:
1) detection 1 (+), detection 2 (+): MTB infect；
2) detection 1 (-), detection 2 (+): NTM infect；
3) detection 1 (-), detection 2 (-): without mycobacterial infections.
[embodiment 2] testing result
Laboratory carries out lunger's (totally 273 example in a small amount, include strain identification result 92 example and only expectorant be coated with positive 85 examples add up to bacterium sun patient 187 examples, and expectorant is coated with bacterium the moon patient 86 example all negative with Sputum culturing) peripheral blood testing inspection, result is as shown in Table 1 and Table 2.
Table 185 example MTB and 7 example NTM infectivity bacterium sun lunger's peripheral blood ELISPOT testing results
Table 295 example expectorant is coated with positive (bacterium sun) pulmonary tuberculosis and 86 example bacterium the moon lunger's peripheral blood ELISPOT testing results
Result above is pointed out, and is no matter bacterium sun or bacterium the moon pulmonary tuberculosis all has caused by the NTM of some ratios infects.Therefore, no matter it is tuberculosis (bacterium sun and bacterium the moon pulmonary tuberculosis, the outer tuberculosis of lung etc.) patient, or LTBI person, if detecting with conventional ELISPOT test kit, it is possible to can lose, reduce the positive rate of part infection detection；And the test kit of the present invention perhaps can make up the deficiency of this respect to a certain extent.
The invention is not limited in above-mentioned embodiment, if to the various changes of the present invention or deformation without departing from the spirit and scope of the present invention, if these are changed and deform within the claim and the equivalent technologies scope that belong to the present invention, then the present invention is also intended to comprise these changes and deformation.
Claims (9)
1. the test kit differentiating that mycobacterium tuberculosis infects with non-tuberculous mycobacteria, it is characterised in that: include the specific antigen of mycobacterium tuberculosis, mycobacteria genus-specific antigen and the detection T cell instrument to described antigen recognition.
2. test kit according to claim 1, it is characterized in that: by utilizing the specific antigen of mycobacterium tuberculosis to contact with ex vivo T-cell to be measured respectively with mycobacteria genus-specific antigen, and detect the cytokine that this T cell discharges, identify that testing sample is mycobacterium tuberculosis or non-tuberculous mycobacteria infection.
3. test kit according to claim 2, it is characterized in that: specific for described mycobacterium tuberculosis antigen and mycobacteria genus-specific antigen testing result are compared, judge that described ex vivo T-cell to be measured is m tuberculosis infection or non-tuberculous mycobacteria infection according to comparative result.
4. test kit according to claim 3, it is characterised in that if the specific antigen of described mycobacterium tuberculosis and the detection of mycobacteria genus-specific antigen are all positive, then described ex vivo T-cell to be measured is m tuberculosis infection；If if the specific Detection of antigen result of described mycobacterium tuberculosis is negative, described mycobacteria genus-specific antigen testing result is positive, then described ex vivo T-cell to be measured is that non-tuberculous mycobacteria infects.
5. test kit according to claim 1, it is characterised in that the specific antigen of described mycobacterium tuberculosis includes one or more in polypeptide E6, E7, C14.
6. test kit according to claim 5, it is characterised in that the instrument of described antigen recognition is IFN-γ antibody.
7. test kit according to claim 5, it is characterised in that the cytokine of the ex vivo T-cell release that the tool detection of described antigen recognition is to be measured.
8. test kit according to claim 7, it is characterised in that described T cell derives from blood, bronchoalveolar lavage fluid, hydrothorax, cerebrospinal fluid, lymph node.
9. the test kit according to any bar in claim 1-8, it is characterised in that described test kit also includes negative control group and positive controls, and described positive controls contains PMA and ionomycin.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610210106.3A CN105807051A (en) | 2016-04-06 | 2016-04-06 | Kit for identifying MTB (mycobacterium tuberculosis) infection and NTM (nontuberculosis mycobacteria) infection |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610210106.3A CN105807051A (en) | 2016-04-06 | 2016-04-06 | Kit for identifying MTB (mycobacterium tuberculosis) infection and NTM (nontuberculosis mycobacteria) infection |
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| CN105807051A true CN105807051A (en) | 2016-07-27 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110702918A (en) * | 2019-09-24 | 2020-01-17 | 广州迪澳医疗科技有限公司 | Kit for rapidly detecting active tuberculosis |
| CN111905110A (en) * | 2020-07-27 | 2020-11-10 | 北京恩元华生物科技有限公司 | Method, system and application for identifying mycobacterium tuberculosis infection and nontuberculous mycobacterium infection |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101413031A (en) * | 2008-12-02 | 2009-04-22 | 博奥生物有限公司 | Method for identifying Mycobacterium tuberculosis and non-tuberculous mycobacteria, and special reagent kit therefor |
| CN101805397A (en) * | 2009-02-18 | 2010-08-18 | 上海市公共卫生临床中心 | Mycobacterium tuberculosis ESAT-6 recombinant dipolymer, preparation method and application thereof |
-
2016
- 2016-04-06 CN CN201610210106.3A patent/CN105807051A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101413031A (en) * | 2008-12-02 | 2009-04-22 | 博奥生物有限公司 | Method for identifying Mycobacterium tuberculosis and non-tuberculous mycobacteria, and special reagent kit therefor |
| CN101805397A (en) * | 2009-02-18 | 2010-08-18 | 上海市公共卫生临床中心 | Mycobacterium tuberculosis ESAT-6 recombinant dipolymer, preparation method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| YI PENG ET AL.: "Interferon-gamma ELISPOT for the screening and diagnosis of latent tuberculosis infection in healthy population of China", 《ADVANCES IN BIOSCIENCE AND BIOTECHNOLOGY》 * |
| 乐军 等: "酶联免疫斑点试验快速诊断结核分枝杆菌感染的临床应用价值", 《中华检验医学杂志》 * |
| 林淑娴 等: "酶联免疫斑点法对抗结核治疗期肺结核患者多肽特异性IFN-γ 的检测", 《中国医药生物技术》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110702918A (en) * | 2019-09-24 | 2020-01-17 | 广州迪澳医疗科技有限公司 | Kit for rapidly detecting active tuberculosis |
| CN111905110A (en) * | 2020-07-27 | 2020-11-10 | 北京恩元华生物科技有限公司 | Method, system and application for identifying mycobacterium tuberculosis infection and nontuberculous mycobacterium infection |
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